FXR is a molecular target for the effects of vertical sleeve gastrectomy

Abstract

Bariatric surgical procedures, such as vertical sleeve gastrectomy (VSG), are at present the most effective therapy for the treatment of obesity, and are associated with considerable improvements in co-morbidities, including type-2 diabetes mellitus. The underlying molecular mechanisms contributing to these benefits remain largely undetermined, despite offering the potential to reveal new targets for therapeutic intervention. Substantial changes in circulating total bile acids are known to occur after VSG. Moreover, bile acids are known to regulate metabolism by binding to the nuclear receptor FXR (farsenoid-X receptor, also known as NR1H4). We therefore examined the results of VSG surgery applied to mice with diet-induced obesity and targeted genetic disruption of FXR. Here we demonstrate that the therapeutic value of VSG does not result from mechanical restriction imposed by a smaller stomach. Rather, VSG is associated with increased circulating bile acids, and associated changes to gut microbial communities. Moreover, in the absence of FXR, the ability of VSG to reduce body weight and improve glucose tolerance is substantially reduced. These results point to bile acids and FXR signalling as an important molecular underpinning for the beneficial effects of this weight-loss surgery.

Figure 1. Unbiased pathway analysis in VSG ileum

Based on a pathway analysis of genes differentially regulated (fold change >=1.5) in the terminal ileum of VSG mice relative to sham-operated, pair-fed (SPF) controls, the following top 5 pathways were significantly enriched: Glutathione mediated detoxification (p=6.00E-8), Nicotine degredation (p=5.64E-4), Metapathway biotransformation (p=4.81E-5), Nuclear receptors in lipid metabolism and toxicity (p=7.18E-5), and Type II interferon signaling (p=1.25E-4). n= 5 per group.

Figure 2. FXR contributes to the maintenance of weight loss following VSG

Both WT and FXR KO mice A) weighed more than 30g and B) carried more than 30% of their weight as fat prior to the surgery. C) WT-VSG mice lose weight and maintain this weight loss, relative to WT-sham controls, whereas D) KO-VSG mice recover the initial weight loss within 5 weeks after surgery, relative to KO-sham controls. E) 11-weeks following surgery, WT-VSG mice carry half the body fat of WT-sham mice whereas the body fat of KO-VSG and KO-sham mice is equivalent. F) 11-weeks following surgery, WT-VSG mice have lost 41% of their pre-surgical body fat, whereas KO-VSG mice exhibit no significant fat loss. Data are shown as mean ± SE. *= p< 0.05, ** = p< 0.01, *** = p< 0.001. For panels A-D, n= 12 WT-sham, 8 WT-VSG, 9 KO-sham, 8 KO-VSG. For panels E-F, n= 12 WT-sham, 9 WT-VSG, 9 KO-sham, 10 KO-VSG.

Figure 3. FXR alters feeding behavior following VSG

A,B) WT mice eat significantly less during the first 3 weeks following VSG vs. sham surgery; this is not recovered at later time points. C,D) KO mice eat significantly less during the first week following VSG vs. sham surgery; beginning at week 4 KO-VSG mice compensate for this initial caloric deficit by consuming more calories than sham-operated controls. E) By 8-weeks following surgery, cumulative food intake by WT-VSG mice was 15% less than WT-sham mice, whereas food intake by KO-VSG and sham-operated mice was equivalent. F) When given the choice among three pure macronutrient diets, WT-VSG mice exhibit a blunted preference for dietary fat relative to carbohydrates and protein. G) Among WT mice, VSG reduces the preference for dietary fat. Moreover, loss of FXR is associated with lack of preference for dietary fat that is not further altered by the surgery. Data are shown as mean ± SE. * = p< 0.05, ** = p< 0.01, *** = p< 0.001. For panels A-E, n= 13 WT-sham, 10 WT-VSG, 9 KO-sham, 10 KO-VSG. For panels F-G, n= 12 WT-sham, 6 WT-VSG, 8 KO-sham, 7 KO-VSG.

Figure 4. FXR contributes to the improvement in glucose tolerance observed following VSG

A) Among WT mice, fasting blood glucose is reduced following VSG, whereas among KO mice it is increased. B-C) Among WT mice, VSG improves the excursion of blood glucose following an i.p. bolus of 1 g/kg dextrose; among KO mice the glucose tolerance of VSG and sham-operated mice is equivalent. Data are shown as mean ± SE. * = p< 0.05, ** = p< 0.01, *** = p< 0.001. n= 13 WT-sham, 10 WT-VSG, 9 KO-sham, 10 KO-VSG.

Figure 5. VSG and FXR alter the composition of cecal microbial communities

A) Principal coordinates analysis (PCoA) plot of weighted UniFrac distances. Each dot represents a cecal community. The percentage of variation explained by each principal coordinate is shown in parentheses. B) Abundance gradient of Bacteroidetes and C) abundance gradient of Firmicutes. Gradients range from blue (indicating low relative abundance) to red (indicating high relative abundance). The same plots are shown for A–C). D) Relative abundance of Bacteroides was reduced in WT-VSG compared to WT-sham operated mice, but did not differ in KO mice E) Relative abundance of one genus in the Porphyromonadaceae family was increased in WT-VSG compared to WT-sham operated mice, but was reduced in KO-VSG mice relative to KO-sham operated controls F) Relative abundance of Roseburia was increased in WT-VSG compared to WT-sham operated mice, but did not differ in KO mice G) Among WT-VSG mice, the relative abundance of Roseburia was tightly correlated with fasting blood glucose. Data are presented as Tukey boxplots. * = p< 0.05, ** = p< 0.01, *** = p< 0.001. n= 12 WT-sham, 7 WT-VSG, 9 KO-sham, 8 KO-VSG.

Extended Data Figure 1. Body weight and body fat in a weight-matched subset of WT and FXR KO mice

These posthoc analyses include the lightest 9 WT and heaviest 8 FXR-KO mice prior to surgery, creating 4 well-matched groups. In this subset, (A) WT-VSG lose weight relative to WT-sham controls, and maintain this weight loss for 14 weeks, whereas (B) KO-VSG mice lose weight initially, but recover to match the weight of KO-sham controls within 4-5 weeks. Likewise (C-D) these groups were well-matched for pre-surgical body fat. At 11 weeks after surgery, WT-VSG mice had significantly less body fat compared to both WT-sham controls and KO-VSG mice. KO-sham and KO-VSG mice had equivalent adiposity. Data are shown as mean ± SE. *= p< 0.05, ** = p< 0.01, *** = p< 0.001. n= 5 WT-sham, 4 WT-VSG, 4 KO-sham, 4 KO-VSG.

Extended Data Figure 2. Glucose tolerance in FXR-VSG and WT-VSG mice

When the glucose excursion of WT-VSG and KO-VSG mice are compared directly, KO-VSG mice exhibit significantly impaired glucose clearance at both 30 and 60 minutes. Data are shown as mean ± SE. *= p< 0.05. n= 10 per group.

Extended Data Figure 3. Effect of genotype and VSG on distribution along PC1

Among WT mice, sham and VSG mice separate significantly along PC1. In contrast, among KO mice there is no significant difference between sham and VSG. Data are shown as mean ± SE. *= p< 0.05. n= 12 WT-sham, 7 WT-VSG, 9 KO-sham, 8 KO-VSG.

Extended Data Figure 4. Relative abundance of Bacteroides, an uncharacterized genus in Porphyromonadaceae, and Roseburia correlated with metabolic parameters

The relative abundance of Bacteroides was significantly correlated with change in body weight (A), change in body fat (B), and the area under the curve (AUC) in the glucose tolerance test (D), but not with fasting blood glucose (C). The relative abundance of an uncharacterized genus in Porphyromonadaceae was significantly correlated with change in body weight (E), fasting blood glucose (G), and the area under the curve (AUC) in the glucose tolerance test (H), but not with change in body fat (F). The relative abundance of Roseburia was significantly correlated with change in body weight (I), change in body fat (J), fasting blood glucose (K), and the area under the curve (AUC) in the glucose tolerance test (L). n=36.

Extended Data Figure 5. Effect of genotype and VSG on the relative abundance of Lactobacillus, Lactococcus, and Escherichia/ Shigella

VSG was associated with a significant increase in the relative abundance of Lactobacillus (A), Lactococcus (B), and Escherichia/ Shigella that did not vary according to genotype. Data are presented as Tukey boxplots. n= 12 WT-sham, 7 WT-VSG, 9 KO-sham, 8 KO-VSG.

Extended Data Figure 6. VSG alters the abundance of cecal SCFAs

The relative concentration of butyrate (A) and propionate (B), but not acetate (C) was altered by VSG, and this did not differ depending on genotype. D) The acetate:butyrate ratio is increased following VSG. Data are presented as Tukey boxplots. * = p< 0.05, ** = p< 0.01, *** = p< 0.001. Also see Extended Data Table1. n= 12 WT-sham, 7 WT-VSG, 9 KO-sham, 8 KO-VSG.